Breathing is a remarkable behavior that mediates gas exchange to support metabolism and regulate pH. A reliable and robust rhythm is essential for breathing movements in mammals. Failure to maintain a normal breathing rhythm in humans suffering from sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. Neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brainstem areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the site(s) and mechanisms for respiratory rhythmogenesis must be revealed. A brainstem region critical for normal breathing is the preBtzinger Complex (preBtC). Studies of respiratory rhyth- mogenesis have focused primarily on preBtC neuron cellular properties or regional interactions between respiratory-related brainstem centers, largely ignoring the (micro)circuit structure within each of these essential areas, including the preBtC. While informative, these prior studies have failed to reveal validated mechanisms underlying rhythmogenesis. We break from current paradigms and propose a completely novel hypothesis: Respiratory rhythm is generated by preBtC burstlets, low level synchronous multineuronal activity, which normally trigger the high amplitude bursts necessary to produce respiratory motor output. To test this burstlet hypothesis, we will determine key cellular, synaptic and network mechanisms underlying burstlets and bursts using a novel protocol in AIM 1. To examine how the (micro)circuit structure contributes to rhythmogenesis, we take advantage of recent technological advances that allow spatiotemporally precise perturbation of neural micro(circuits) in AIM 2. We utilize an advanced optical technique, holographic photolysis, to map connections within the preBtC and determine network interactions underlying respiratory rhythmogenesis. Breathing is a rhythmic behavior of fundamental importance. By determining the mechanisms underlying the generation of respiratory rhythm, we will significantly improve our knowledge of neural control over the entire breathing cycle. These studies should make fundamental contributions to our understanding of breathing in humans in health and disease.